Process for the production of fruit polyphenols from unripe rosaceae fruit

ABSTRACT

The present invention provides a fruit polyphenol obtained by subjecting unripe fruits of Rosaceae to pressing and/or extraction and then purifying the resulting juice or extract. The present invention further provides an antioxidant, a hypotensive agent, an antimutagenic agent, an antiallergic agent and an anticariogenic agent each comprising, as an effective component, a fruit polyphenol obtained by subjecting unripe fruits of Rosaceae to pressing and/or extraction and then purifying the resulting juice or extract. The fruit polyphenol of the present invention has various physiological activities, for example, an antioxidative activity, an ACE-inhibiting activity, an antimutagenic activity, a hyalulonidase-inhibiting activity and a GTase-inhibiting activity.

This application is a continuation of application Ser. No. 08/278,080filed Jul. 20, 1994, now abandoned.

BACKGROUND OF THE INVENTION AND THE RELATED ART

The present invention relates to a fruit polyphenol; a process forproduction thereof; and an antioxidant, a hypotensive agent, anantimutagenic agent, an antiallergic agent and an anticariogenic agenteach comprising said polyphenol as an effective component.

In growing the trees of Rosaceae which bear edible fruits such as apple,pear, peach and the like, "thinning-out" of superfluous fruits isgenerally carried out in the period of mid May to mid July. In thisthinning-out, unripe fruits in the form of bunch or cluster are removedwith some of them left unremoved. Consequently, a large amount of unripefruits are disposed by the thinning-out, without being utilized. Theseunripe fruits are very bitter as compared with ripe fruits, and thesectional surface of each unripe fruit turns brown easily. This factsuggests the presence of a large amount of polyphenol compounds inunripe fruits.

It is known that polyphenol compounds are generally present in the plantkingdom as a secondary metabolite of plant in numerous kinds and in alarge amount. Some of these polyphenol compounds have drawn attentionfor their diversified physiological activities, from the past in thefield of pharmacology and in recent years in the field of foodchemistry.

Among them, tea polyphenol (catechins) is drawing particular attentionand concentrated researches are under way thereon. This tea polyphenolis being recognized to have very wide physiological activities such asantibacterial activity, antiviral activity, antioxidative activity,antimutagenic activity, anticancerous activity, plateletcoagulation-inhibiting activity, blood pressure increase-inhibitingactivity, blood sugar increase-inhibiting activity, bloodcholesterol-reducing activity, anticariogenic activity, antiallergicactivity, intestinal flora-improving activity, deodoring activity andthe like [Japanese Patent Application Kokai (Laid-Open) Nos.214183/1988, 6499/1990, 178320/1992, etc.].

Thus, it is known that, for example, the polyphenol extracted from teahas wide physiological activities.

SUMMARY OF THE INVENTION

An object of the present invention is to provide a fruit polyphenolhaving various physiological activities, which is different from thepolyphenol extracted from tea.

Another object of the present invention is to provide a process forproducing the above fruit polyphenol effectively and economically.

According to the present invention, there is provided a fruit polyphenolobtained by subjecting unripe fruits of Rosaceae, particularly unripeapples, unripe pears or unripe peaches to pressing and/or extraction andthen purifying the resulting juice or extract.

According to the present invention, there is further provided a processfor producing a fruit polyphenol, which comprises subjecting unripefruits of Rosaceae, particularly unripe apples, unripe pears or unripepeaches to pressing and/or extraction and then purifying the resultingjuice or extract to obtain a polyphenol fraction.

According to the present invention, there is further provided anantioxidant comprising, as an effective component, a fruit polyphenolobtained by subjecting unripe fruits of Rosaceae, particularly unripeapples, unripe pears or unripe peaches to pressing and/or extraction andthen purifying the resulting juice or extract.

According to the present invention, there is further provided ahypotensive agent comprising, as an effective component, a fruitpolyphenol having an inhibitory activity for angiotensin convertingenzyme I, which polyphenol is obtained by subjecting unripe fruits ofRosaceae, particularly unripe apples, unripe pears or unripe peaches topressing and/or extraction and then purifying the resulting juice orextract.

According to the present invention, there is further provided anantimutagenic agent comprising, as an effective component, a fruitpolyphenol having an activity capable of inhibiting the mutagenicity ofcarcinogenic substance, which polyphenol is obtained by subjectingunripe fruits of Rosaceae, particularly unripe apples, unripe pears orunripe peaches to pressing and/or extraction and then purifying theresulting juice or extract.

According to the present invention, there is further provided anantiallergic agent comprising, as an effective component, a fruitpolyphenol having a hyaluronidase-inhibiting activity, which polyphenolis obtained by subjecting unripe fruits of Rosaceae, particularly unripeapples, unripe pears or unripe peaches to pressing and/or extraction andthen purifying the resulting juice or extract.

According to the present invention, there is further provided ananticariogenic agent comprising, as an effective component, a fruitpolyphenol having a glucosyltransferase-inhibiting activity, whichpolyphenol is obtained by subjecting unripe fruits of Rosaceae,particularly unripe apples, unripe pears or unripe peaches to pressingand/or extraction and then purifying the resulting juice or extract.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is graphs showing the HPLC chromatogram and the comparison of UVspectle of each peaks of unripe apple polyphenol.

FIG. 2 is graphs each showing the relation between the amount ofperoxylipid formed (the absorbance at 500 nm) and the additionconcentration of extract from unripe or ripe apple "Fuji", in one weekafter said extract was added to a reaction system.

FIG. 3 is graphs each showing the relation between the amount ofperoxylipid formed (the absorbance at 500 nm) and the additionconcentration of polyphenol fraction of unripe or ripe apple "Fuji", inone week after said fraction was added to a reaction system.

FIG. 4 is graphs showing the amounts of peroxylipids formed (theabsorbances at 500 nm) in one week after various polyphenol compoundswere added to respective reaction systems.

FIG. 5 is graphs each showing the relation between the amount ofperoxylipid formed (the absorbance at 500 nm) and the additionconcentration of polyphenol fraction of each unripe apple, in one weekafter said fraction was added to a reaction system.

FIG. 6 is graphs each showing the relation between the amount ofperoxylipid formed (the absorbance at 500 nm) and the additionconcentration of polyphenol fraction of each unripe fruit, in one weekafter said fraction was added to a reaction system.

FIG. 7 is graphs each showing the angiotensin I converting enzyme(hereinafter referred to as ACE)-inhibiting activity of polyphenolfraction of unripe or ripe apple "Fuji".

FIG. 8 is graphs showing the ACE-inhibiting activities of variouspolyphenol fractions B when each polyphenol fraction B was added to areaction system in various amounts.

FIG. 9 is graphs showing the ACE-inhibiting activities of variouspolyphenol compounds when each polyphenol compound was added to areaction system in various amounts.

FIG. 10 is a graph showing the absorption spectrum of an isolatedpolyphenol fraction, measured by a spectrophotometer.

FIG. 11 is graphs showing the results of measurement by GPC, ofmolecular weights of condensed tannin.

FIG. 12 is the structural formula of the ACE-inhibiting substancecontained in the fruit polyphenol of the present invention.

FIG. 13 is graphs showing the antimutagenic activities of variouspolyphenols to Trp-P-2.

FIG. 14 is graphs showing the antimutagenic activities of apple tanninand epigallocatechin gallate to benzo[a]pyrene.

FIG. 15 is graphs showing the hyaluronidase-inhibiting activities ofvarious fruit extracts.

FIG. 16 is graphs showing the hyaluronidase-inhibiting activities of anantiallergic agent (sodium cromoglicate) and apple tannin.

FIG. 17 is graphs showing the relations between the amount of insolubleglucan formed and the amount of fruit polyphenol fraction added, whenvarious fruit polyphenol fractions were added to respective reactionsystems.

FIG. 18 is graphs showing the relations between the amount of insolubleglucan formed and the amount of polyphenol compound added, when variouspolyphenol compounds were added to respective reaction systems.

DETAILED DESCRIPTION OF THE INVENTION

The fruit polyphenol according to the present invention is a polyphenolobtained by subjecting unripe fruits of Rosaceae, particularly unripeapples, unripe pears or unripe peaches to pressing or extraction andthen purifying the resulting juice or extract. The purification isconducted by treating the juice or extract with an adsorbent, and thefraction of the juice or extract adsorbed by the adsorbent (thisfraction is hereinafter referred to as "adsorbed fraction") contains afruit polyphenol. The adsorbed fraction is eluted with an anhydrousalcohol (e.g. ethanol) to obtain a purified polyphenol fraction.

This polyphenol fraction is then concentrated, whereby a liquidpolyphenol product can be obtained.

When the concentrate obtained above is subjected to spray-drying orfreeze-drying, a powder polyphenol product can be obtained.

The starting material used in the present invention is fruits ofRosaceae. Specifically, apples, pears, peaches, etc. are preferable withapples being particularly preferable. The fruits may be ripe or unripe,but unripe fruits are particularly preferable because they containpolyphenol compounds in a larger total amount and also because theycontain various polyphenol compounds having diversified physiologicalactivities, in large amounts.

Description is made on the pressing. In one pressing method, thestarting material is washed; the washed material is subjected tocrushing and pressing with or without addition of sulfurous acid, toobtain a juice; preferably, a pectolytic enzyme is added thereto; andthe resulting mixture is subjected to centrifugation, filtration or thelike to obtain a clear juice.

Description is made on the extraction. In one extraction method, thewashed material is mixed with an alcohol (e.g. ethanol or methanol); themixture is subjected to crushing; the resulting material is subjected toextraction while it is being immersed and pressed, or refluxed; theextract is concentrated under reduced pressure to remove the alcohol;and the concentrate is subjected to centrifugation and filtration, or todistribution with an organic solvent (e.g. hexane or chloroform) andfiltration, to obtain a clear extract.

Description is made on the purification of the juice or extract obtainedabove. The clear juice or clear extract obtained above is passed througha column filled with an adsorbent capable of selectively adsorbing thefruit polyphenol contained in the juice or extract and also capable ofreleasing the adsorbed polyphenol by the use of an elutant, whereby apolyphenol fraction is adsorbed. [Examples of the adsorbent are astyrene-divinylbenzene type synthetic resin, an anion exchange resin andan octadecyl group-chemically bonded silica gel (ODS)]. Then, distilledwater is passed through the column for washing. Thereafter, a 20-100%alcohol (e.g. ethanol) solution, preferably an about 50% alcoholsolution is passed through the column, whereby a polyphenol fraction iseluted and recovered. The resulting polyphenol solution is concentratedunder reduced pressure to remove the alcohol, whereby a fruit polyphenolliquid product (preferably, an organic acid such as malic acid or thelike is added therein) can be obtained. This liquid product is subjectedto spray-drying or freeze-drying, after addition or no addition of anauxiliary agent for powdering such as dextrin or the like, whereby afruit polyphenol powder product can be obtained.

According to the confirmation by the present inventor, the fruitpolyphenol obtained by the present invention is composed mostly of (1)simple polyphenol compounds such as caffeic acid derivatives, p-coumaricacid derivatives, flavan-3-ols (catechins), flavonols (quercetinglycosides), dihydrochalcones (phloretin glycosides) and the like and(2) high-molecular polyphenol compounds such as condensed tannins andthe like.

Thus, the fruit polyphenol obtained in the present invention isconsidered to have various physiological functions, and the presentinventor made an extensive study. As a result, it was first found thatthe fruit polyphenol of the present invention contains a large amount ofa component capable of inhibiting the oxidation of linoleic acid (avegetable oil). Therefore, the fruit polyphenol of the present inventionis very effective as an antioxidant.

It was next found that the fruit polyphenol of the present inventioncontains a large amount of a component capable of inhibiting thefunction of ACE (this is an enzyme connected with an increase in bloodpressure). Therefore, the fruit polyphenol of the present invention isvery effective also as a hypotensive agent.

A further study was made to identify the ACE-inhibiting componentcontained in the fruit polyphenol of the present invention. As a result,the ACE-inhibiting component was confirmed to be a condensed tanninrepresented by the structural formula shown in FIG. 12.

Many researches conducted in recent years indicated that there is a highcorrelation between carcinogenic substance and mutagenic substance andaccordingly there is a deep connection between carcinogenicity andmutagenicity. Consequently, a substance capable of inhibitingmutagenicity is expected to be able to prevent carcinogenicity. Thepresent inventor examined whether or not the fruit polyphenol of thepresent invention has an antimutagenic activity, and confirmed that thepolyphenol has said activity. Therefore, the fruit polyphenol of thepresent invention is very effective also as an antimutagenic agent.

Hyaluronidase is an enzyme which is considered to exhibit a certain cellactivity in the reconstruction of connective tissue. Researchesconducted in recent years (Chem. Pharm. Bull., 33, p. 642, 1985; ibid.,33, p. 5079, 1985; ibid., 33, p. 3787, 1985; ibid., 33, p. 5079, 1985;and ibid., 40, p. 1439, 1992) indicated that in synthetic antiallergicagents (e.g. sodium cromoglicate and tranilast) there is a highcorrelation between the hyaluronidase-inhibiting activity and theactivity for suppressing the release of histamine from mast cells.Utilizing this correlation, several antiallergic substances were foundin natural products by measuring said products forhyaluronidase-inhibiting activity. Hence, the present inventor examinedwhether or not the fruit polyphenol of the present invention has ahyaluronidase-inhibiting activity, and confirmed that the polyphenol hassaid activity. Therefore, the fruit polyphenol of the present inventionis very effective also as an antiallergic agent.

Dental caries (decayed tooth) is currently confirmed to be abacteria-related disease caused by oral Streptococcus includingStreptococcus mutans. In the process of dental caries development,formation of deposit is considered to be a particularly importantfactor.

That is, there is synthesized, from the sucrose contained in foods,sticky and insoluble glucan by the action of glucosyltransferase(hereinafter referred to as "GTase") which is an enzyme produced bycariogenic bacteria; bacteria adhere on the dentin via said glucan; andthe resulting deposit causes dental caries.

Thus, substances capable of inhibiting GTase's action are expected to beeffective as an anticariogenic agent [Appl. Env. Microbiol., 59 (4), pp.968-973, 1993; Biosci. Biotech. Biochem., 56 (5), pp. 766-768, 1992;Agric. Biol. Chem., 54 (11), pp. 2925-2929, 1990; and Chem. Pharm.Bull., 38 (3), pp. 717-720, 1990].

Hence, the present inventor examined whether or not the fruit polyphenolof the present invention has an inhibitory activity to insolubleglucan-producing GTase which is produced by S. sobrinus (a typicalcariogenic bacteria), and found that the polyphenol has said activity.Therefore, the fruit polyphenol of the present invention is veryeffective also as an anticariogenic agent.

Next, the present invention is described in more detail referring toExamples. However, the present invention is not restricted to theseExamples.

EXAMPLE 1

Compositional analysis of unripe apple juice

The following samples were subjected to general compositional analysis.

Samples

Ripe apple "Fuji": commercial product grown using no bag

Unripe apple "Fuji": collected in mid June

Treatment of samples

Each fruit sample was crushed by means of a mixer, with an appropriateamount of potassium metabisulfite (an antioxidant) being added. Theresulting juice was subjected to centrifugation and filtration to obtaina clear juice. Each clear juice was measured for the following items.

Measurement items (and methods)

x Average weight of individual fruits (n=50)

x Ratio of juice obtained (%)

x pH

x Acidity (g/l in terms of malic acid)

x Brix

x Total phenols (ppm in terms of chlorogenic acid)

x Total ascorbic acid

x Analysis of organic acids

x Analysis of saccharide

x Analysis of metal ions

x Analysis of free amino acids

The results of measurements are shown in Table 1.

    TABLE 1       -  Average weight of Ratio of juice  Acidity(g/l in terms  Total     phenols(ppm in terms Total ascorbic        individual fruits(g) obtained(%) pH of malic acid) Brix of chlorogenic       acid) acid(ppm)       Ripe "Fuji" juice 287.2 66.1 3.88 2.73 11.6      720 --                    Unripe "Fuji" juice  5.1 51.0 3.78 6.83  5.2     6,200 254       Metal ions (ppm)        K Ca Mg Fe Na       Ripe "Fuji" juice 1,430  15  16  0      9                                   Unripe "Fuji" juice 3,050 250 130     13 28       Organic acids (%) Saccharide (%)        Malic Quinic        acid acid (Total) Glucose Fructose Sucrose Sorbitol (Total)       Ripe "Fuji" juice 0.39 0.02 (0.41) 2.12 6.72 2.52 0.31 (11.67)              Unripe "Fuji" juice 0.49 1.17 (1.65) 0.70 0.49 0.03 0.44     (1.66)       Free amino acids        ASP THR SER ASN GLU GLN GLY ALA CIT VAL MET ILEU LEU TYR PHE α      ABA γ      ABA MEA HYLYS 1M-HIS HIS LIS ARG       Ripe "Fuji" juice 211  2 10   207 29  2   4 3  2   7      3             Unripe "Fuji" juice  32 40 42 3,280  13 36 52 4 37 8 25     52 76 114  337 44 9 14 19 47 34

As is clear from Table 1, there is a big difference in compositionbetween unripe apple juice and ripe apple juice. In the unripe applejuice were abundant, in particular, total phenols, total ascorbic acid,organic acids (particularly, quinic acid), metal ions and free aminoacids (asparagine, phenylalanine and γ-aminobutyric acid, inparticular). Meanwhile, saccharide was very little in the unripe applejuice.

Next, the polyphenol contained in unripe apple was subjected tocompositional analysis by HLPC.

As the polyphenol sample, there was used a purified polyphenol obtainedby subjecting an unripe apple extract to solid-phase extraction (seeExample 2).

The results of the analysis is shown in FIG. 1.

As is clear from FIG. 1, the sample polyphenol contained in unripe applewere composed mostly of caffeic acid derivatives (chlorogenic acid,etc.), p-coumaric acid derivatives, flavan-3-ols (catechin, epicatechin,etc.), flavanols (quercetin glycosides) and dihydrochalcones (phloretinglycosides, particularly phloridzin). Among them, chlorogenic acid,catechin, epicatechin and phloridzin were contained in large amounts.

EXAMPLE 2

Antioxidative activities of polyphenols in unripe fruits

Starting materials: the following starting materials were used.

Ripe apple: commercial product grown using no bag

Unripe apples: collected in mid June

"Fuji": average weight=4.97 g (n=50)

"Tugaru": average weight=7.80 g (n=50)

"Jonagold": average weight=3.86 g (n=50)

"Hokuto": average weight=3.32 g (n=50)

"Ohrin": average weight=10.34 g (n=50)

Unripe pear: collected in early June

"Hohsui": average weight=8.98 g (n=50)

Unripe peach: collected in early June

"Akatuki": average weight=5.03 g (n=50)

Preparation of samples

Juice samples: the Example 1 procedure was repeated to obtain clearjuices.

Extract samples: each extract sample was obtained by homogenizing 400 gof a starting material together with 1% HCl-containing methanol,subjecting the resulting material to extraction (three times) withrefluxing, concentrating the extract under reduced pressure to removemethanol, adding chloroform thereto to conduct distribution (two times),recovering the aqueous layer, filtering the aqueous layer, and thenadding distilled water to the filtrate to make the total volume 200 ml.

As necessary, the juice samples and the extract samples were subjectedto solid-phase extraction using Sep-pack C18 to obtain polyphenolfractions.

Test method for antioxidative activity

The following test method for antioxidative activity was employed.

5 ml of a base solution (ethanol containing 4% of linolic acid) wasmixed with 4 ml of a phosphate buffer solution (pH=7.0) and 1 ml of asample solution in a tightly stoppered test tube. The test tube was keptin a 50° C. thermostat under light shielding. Also kept under the sameconditions were a test tube (control) containing ethanol in place oflinolic acid and a test tube (blank) containing the sample solvent inplace of the sample.

During the storage period, the reaction mixture was sampled with thelapse of time to quantitatively determine the amount of peroxide formedby the isothiocyanate method (the iron rhodanide method).

Results of measurements

Antioxidative activity was examined on the unripe apple "Fuji" extractand the ripe apple "Fuji" extract.

The above two extracts were each added to a reaction system in aconcentration range of 1-1,000 μl/g (linolic acid). One weak later theaddition, the amount of peroxylipid formed (absorbance at 500 nm) wasmeasured. Its correlation with the concentration of extract added isshown in FIG. 2.

As is shown from FIG. 2, the antioxidative activity to linolic acid isseen in both the unripe "Fuji" extract and the ripe "Fuji" extract, butthe activity is higher in the unripe "Fuji" extract.

Next, in order to investigate what the substances of antioxidativeactivity were, the unripe "Fuji" extract and the ripe "Fuji" extractwere each largely divided into two fractions, i.e. a fraction A (anon-adsorbed fraction) and a fraction B (an adsorbed fraction, apolyphenol fraction), using Sep-pack C18. Each extract, each fraction Aand each fraction B were independently added to a reaction system in aconcentration range of 1-1,000 μl/g (linolic acid). One week after theaddition, the amount of peroxylipid formed (absorbance at 500 nm) wasmeasured. Its correlation with the amount of extract or fraction A or Bis shown in FIG. 3.

It is clear from FIG. 3 that the antioxidative activity of the ripeapple "Fuji" extract is based on the fraction B of the extract. In theunripe apple "Fuji" extract, both of the fractions A and B haveantioxidative activity but the activity of the fraction B is higher.

From the above, it is indicated that the antioxidative activities of theripe and unripe apple "Fuji" extracts are mostly based on the compoundspresent in the fractions B, i.e. the polyphenol compounds present in thefractions B.

Of the simple polyphenol compounds contained in apples, those compoundsof commercial availability were measured for antioxidative activity.Each of such compounds was added to a reaction system in a concentrationof 2 μM/g (linolic acid). One week after the addition, the amount ofperoxylipid formed (absorbance at 500 nm) was measured. The absorbancesin one week after various polyphenol compounds were added, are shown inFIG. 4.

In FIG. 4, there are included, besides the above polyphenols,comparative substances, i.e. three known antioxidants (BHA, BHT andvitamine E) and typical compounds present in the above fractions A[quinic acid, malic acid and γ-aminobutyric acid (GABA)].

As a result, of the polyphenol compounds, six compounds [caffeic acid,chlorogenic acid, (+)-catechin, (-)-epicatechin, quercetin and rutin(quercetin-3-rha) had antioxidative activities equivalent to those ofBHA and BHT. However, polyphenols such as p-coumaric acid, phloretin andphloridzin had no antioxidative activity. Further, no compounds presentin the fractions A had any antioxidative activity.

By the comparison between (1) the amounts of the compounds present inapple and (2) their antioxidative activities, it could be judged thatthe high antioxidative activity exhibited by the unripe apple extractand the unripe apple juice is given mostly by chlorogenic acid,(+)-catechin and (-)-epicatechin.

From the above, it was confirmed that unripe apple "Fuji" had a highantioxidative activity. Further, the compounds showing said activity,present in the unripe apple "Fuji" were identified.

Successively, it was also investigated whether or not unripe applesother than unripe "Fuji" had the same antioxidative activity.

From the extracts of various unripe apples ("Fuji", "Tugaru","Jonagold", "Hokuto" and "Ohrin") were prepared respective fractions B(polyphenol fractions). Each fraction was added to a reaction system ina concentration range of 1-500 μl/g (linolic acid). One week after theaddition, the amount of peroxylipid formed (absorbance at 500 nm) wasmeasured. Its correlation with the concentration of fraction added isshown in FIG. 5. In FIG. 5, the result of unripe "Fuji" is also includedfor comparison.

As a result, each apple kind showed a high antioxidative activity andthere was substantially no difference in antioxidative activity betweenapple kinds.

Further, the antioxidative activities of unripe fruits other than unripeapples were investigated.

From the extracts of various unripe fruits (apple "Fuji", pear "Hohsui"and peach "Akatuki") were prepared respective fractions B (polyphenolfractions). Each fraction was added to a reaction system in aconcentration range of 1-500 μl/g (linolic acid). One week after theaddition, the amount of peroxylipid formed (absorbance at 500 nm) wasmeasured. Its correlation with the concentration of fraction added isshown in FIG. 6.

As a result, the unripe peach had an antioxidative activity about equalto that of the unripe apple, and the unripe pear had a fairly highantioxidative activity although the activity was inferior to that of theunripe apple.

EXAMPLE 3

ACE (angiotensin I converting enzyme)-inhibiting activities ofpolyphenols present in unripe fruits

Starting materials: the following starting materials were used.

Unripe apples: the same as used in Example 2

Preparation of samples: "extract samples" and "polyphenol fractions"were prepared in the same manner as in Example 2.

Test method for ACE-inhibiting activity

The test for ACE-inhibiting activity was conducted by an ordinarymethod.

That is, a sample solution was added to a commercial ACE solution toconduct preincubation; then, Bz-Gly-His-Leu was added as a substrate togive rise to a reaction; the His fragment formed by the reaction waslabelled with orthophthaldialdehyde; and then the fluorescent intensity(Ex. 360 nm, Em. 490 nm) of the resulting solution was measured.

The ACE-inhibiting activity of the sample solution was expressed by thefollowing formula:

    {1-(S-S.sub.B)/(C-C.sub.B)}×100 (%)

where S: the fluorescent intensity of test solution,

C: the fluorescent intensity of comparative solution wherein water wasused in place of the sample,

S_(B) : the fluorescent intensity of blank for S (the blank containedwater in place of the enzyme), and

C_(B) : the fluorescent intensity of blank for C (the blank containedwater in place of the enzyme).

Results of measurements

Unripe apple "Fuji" extract and ripe apple "Fuji" extract werefractionated into respective unadsorbed fractions (fractions; A) andpolyphenol fractions (fractions B), using Sep-pak C18. Each fraction wasmeasured for ACE-inhibiting activity. The results are shown in FIG. 7.

As a result, in both of the unripe and ripe apples, the fractions Bshowed high ACE-inhibiting activities. In particular, the fraction B ofthe unripe apple exhibitied 100% inhibition. In order to compare theACE-inhibiting activities of the fractions B of the unripe and ripeapples in more detail, there were examined the changes of theACE-inhibiting activities of the two fractions B when the amount of eachfraction B added to the above reaction system was varied. The resultsare shown in FIG. 8.

As a result, the concentration showing 50% inhibition, i.e. IC₅₀ of thefraction B of the ripe apple was about 3 μl. In contrast, the IC₅₀ ofthe fraction B of the unripe apple was 0.1 μl or below. Thus, thefraction B of the unripe apple showed a very high ACE-inhibitingactivity.

This high ACE-inhibiting activity of unripe apple was also observed invarious apples other than "Fuji".

As seen from the above, unripe apples contain compounds having a highACE-inhibiting activity, and the compounds are polyphenols.Incidentally, the ACE-inhibiting activities of unripe pear and unripepeach were lower than those of unripe apples.

Next, ACE-inhibiting activity was measured on each of simple polyphenolspresent in unripe apples and easily available as pure products.Specifically, there were examined the changes of the ACE-improvingactivities of such polyphenls when each polyphenol was added to areaction system in various concentrations. The results are shown in FIG.9.

FIG. 9 includes, for comparison, the ACE-inhibiting activities of teacatechins (tea catechins are known to have ACE-inhibiting activities).In FIG. 9, the IC₅₀ values of (-)-epigallocatechin gallate (EGCg) and(-)-epicatechin gallate (ECg) are 0.3 mM and 2 mM, respectively, andthey have high ACE-inhibting activities. It is reported thatGABALONG-tea (a trade name by Japanese tea marketer), in which the GABAconcentration is increased by processing, showed a hypotensive activityin a test using SHRs (spontaneously hypertensive rats), but GABA showedno ACE-inhibiting activity in the present test.

Meanwhile, the polyphenols present in unripe apple showed ACE-inhibitingactivities when they were at high concentrations. However, their IC₅₀values were low and even in quercetin, which showed the highestactivity, the IC₅₀ was as low as about 5 mM. Thus, the unripe applepolyphenols showed far-lower ACE-inhibiting activities than EGCg andECg.

Hence, based on an assumption that all the polyphenols present in thefraction B of unripe apple are EGCg, the total polyphenol amount presentin the fraction B was calculated using the calibration curve of EGCg. Itwas about 15,000 ppm and the polyphenol concentration in the fraction Bwas calculated to be 33.82 mM in terms of EGCg. Using this calculationvalue, the activity curve of the fraction B was plotted on FIG. 9, whichindicated that the fraction B had a higher ACE-inhibiting activity (IC₅₀=0.2 mM) than EGCg. In actuality, the fraction B contains neither EGCgnor ECg and, as mentioned above, the simple polyphenols in unripe appleshow no striking ACE-inhibiting activity. The high ACE-inhibitingactivity of the unripe apple fraction B observed in FIG. 9 is thought tobe owing to the co-presence of other unidentified polyphenols in thefraction B.

EXAMPLE 4

Identification of ACE-Inhibiting Compounds Present in Unripe ApplePolyphenols

Sample

An unripe apple "Fuji" extract was obtained in the same manner as inExample 3, and the extract was subjected to solid-phase extraction toobtain a polyphenol fraction. This polyphenol fraction was used as asample in the following.

Test method

A sample solution was fed into a Sephadex LH-20 column. The column waswashed with distilled water. Then, elution was conducted with 20-60%methanol (acidificed with HCl) and 70% acetone (acidified with HCl) inthis order to obtain polyphenol fractions.

The obtained polyphenol fractions were each subjected to compositionalanalysis by HPLC, measurement of total phenols and test forACE-inhibiting activity. As necessary, there were also conductedmeasurement of absorption spectrum and gel permeation chromatography.

Test results

Fractionation by the use of Sephadex LH-20 column was tried in order toseparate the ACE-inhibiting compounds in sample from other polyphenols.The fractions obtained by elution were subjected to compositionalanalysis of simple polyphenols by HLPC as well as to ACE-inhibitingtest. All the simple polyphenols were eluted when 60% methanol as anelutant was passed through the column. Meanwhile, all the ACE-inhibitingcompounds were present in the next elutant, i.e. 70% acetone.

The ACE-inhibiting compounds fraction obtained above was subjected todistillation to remove the solvent. The residue was freeze-dried toobtain ACE-inhibiting compounds as a powder. The compounds were readilysoluble in water (the solution was orange) and the yield from 100 ml ofthe polyphenol fraction (fraction B) was about 0.7 g.

The above powder was dissolved in water and the solution was measuredfor absorption spectrum by spectrophotometer. The results are shown inFIG. 10.

The obtained spectrum showed a maximum absorption at 280 nm and had ashape similar to that of (+)-catechin or (-)-epicatechin. The abovesolution was subjected to an autoclvae treatment at 120° C. for 10minutes. In the treatment, the solution turned red and anthocyanidin isthought to have been formed. These results strongly suggest that theabove ACE-inhibiting compounds are procyanidins (regular polymers ofcatechins) known as condensed tannins. In view of the elution behaviorin the Sephadex LH-20 column and the elution behavior in HPLC, saidcompounds are estimated to be not polymers of dimers or so (e.g.procyanidin B₂) but higher polymers.

Hence, the ACE-inhibiting compounds were subjected to molecular weightmeasurement by GPC. It is generally thought that the molecular weightmeasurement of polyphenol compounds by GFC (gel filtrationchromatography) in an aqueous system (GFC in an aqueous system isgenerally used for proteins, etc.) is difficult because polyphenolcompounds exhibit strong affinity with a filler in column, in the formof hydrophobic bond or hydrogen bond. Therefore, the phenolic hydroxylgroup in each ACE-inhibiting compound was acetylated withpyridine/acetic anhydride so that each resulting material became solublein organic solvents, and the resulting materials were subjected to GPCanalysis in an organic solvent (THF). The results are shown in FIG. 11.

In FIG. 11, a single broad peak appeared on the chromatogram. By usingthe molecular weight calibration curve simultaneously prepared usingpolystyrene, the average molecular weight of the ACE-inhibitingcompounds was calculated to be about 2,000.

The structural formula of the ACE-inhibiting compounds was tentativelyestimated considering also the results of other tests not explainedherein (e.g. partial decomposition using toluene-α-thiol, FAB-MSmeasurement), and is shown in FIG. 12.

From the above, the ACE-inhibiting compounds are judged to belong tocondensed tannins.

The polyphenol fraction and the 70% acetone elute, i.e. the condensedtannins fraction obtained by subjecting the polyphenol fraction toSephadex LH-20 fractionation were measured for amount of total phenols,and the two amounts were compared. The results are shown in Table 2.

                                      TABLE 2                                     __________________________________________________________________________              Amount of total phenols (ppm in terms of catechin)                            Before fractionation                                                                   After fractionation                                                                    Before fractionation (%)                            (Total polyphenols) (condensed tannins) After fractionation                 __________________________________________________________________________    Unripe apple extract                                                                    22,800   10,800   47.3                                                Ripe apple extract  1,340   770 57.5                                        __________________________________________________________________________

It is clear from Table 2 that the condensed tannins occupy about half ofthe total polyphenols present in unripe apple and are the components oflargest amount present in said polyphenols.

The IC₅₀ of the condensed tannins to ACE was calculated, and it was verylow (about 1/10 or below of that of EGCg on weight basis). Thus, theACE-inhibiting compounds of the present invention had a very highACE-inhibiting activity.

From the above results, it was judged that the unripe apple polyphenolcan be used as an effective ACE-inhibiting agent.

EXAMPLE 5

Production of unripe fruit polyphenol

About 50 kg of unripe apples (5-10 g/apple) were crushed using a crusherwhile adding an appropriate amount of SO₂, and then pressed using an oilpress. To the resulting juice was added about 50 ppm of a pectolysisenzyme,. and the mixture was subjected to centrifugation or filtrationusing diatomaceous earth and further to precision micro filtration toobtain 35 l of a clear juice. The clear juice was passed through acolumn filled with an industrial use synthetic adsorbent resin (6 l) ofstyrene-divinylbenzene type. Then, 6 l of 0.1% HCl-containing water waspassed through the column to remove saccharide. Thereafter, 0.1%HCl-containing 50% ethanol was passed through to obtain 3 l of afraction containing main polyphenols.

The fraction was concentrated under reduced pressure using anevaporator, to obtain 1.5 l of a concentrated fraction. The concentratedfraction was dried using a spray drier to obtain 228.2 g of an unripeapple polyphenol powder product.

The recovery (%) data are as follows.

Recovery in column: 95.6%

Recovery in spray drying: 93.0%

Recovery from juice: 0.65%

Powder recovery from polyphenols in juice: 88.9%

EXAMPLE 6

Antimutagenic activities of unripe fruit polyphenols

In this Example, antimutagenic activity was measured by modifying theames method (Mutation Research, vol. 31, p. 347, 1975).

Starting materials and preparation of samples

The same as used in Examples 2, 3 and 4. The condensed tannins obtainedin Example 4 are hereinafter referred to as "apple tannin".

Test method for antimutagenic activity

An antimutagenic compound [benzo(a)pyrene or Trp-P-2] solution was mixedwith a phosphate buffer solution, a sample solution, S 9-mix and asalmonella (a solution obtained by culturing Salmonella typhimurium TA98 or TA 100 overnight). The mixture was cultured at 37° C. for 2 days.The number of the resulting colonies was counted. The amount of eachpolyphenol used was shown based on a scale expressed in geometricprogression, between 1 and 300 μg/plate. Antimutagenic activity wascalculated using the following formula:

    antimutagenic activity (%)={(C-B)-(S-B)}÷(C-B)×100

wherein S: number of colonies of test solution

C: number of colonies of control wherein water is used in place of thesample, and

B: number of colonies of blank wherein water is used in place of thesample and the mutagenic compound.

Results

Of various polyphenols and apple tannin, their antimutagenic activites(%) to Trp-P-2 and benzo[a]pyrene are shown in FIG. 13 and FIG. 14,respectively. In each of FIGS. 13 and 14, the axis of ordinate showsantimutagenic activity (%), and the same antimutagenicity as shown inblank was taken as 100%. The axis of abscissa shows the amount of sampleper plate.

As a result, apple tannin inhibited the mutagenicity of each mutagendependently of the concentration of apple tannin, and its inhibitioneffect was equivalent to or even higher than that of epigallocatechingallate. In the test of the present Example, apple tannin showed 50%antimutagenic activity to 1 μg of Trp-P-2, in an amount of about 17 μg(about 51 μg in the case of epigallocatechin gallate), and to 5 μg ofbenzo[a]pyrene in an amount of about 37 μg (about 56 μg in the case ofepigallocatechin gallate). That is, apple tannin contained a largeamount of compounds which strongly inhibitied the mutagenicity ofmutagen (carcinogen). Thus, the fruit polyphenol obtained in the presentinvention is very effective also as an antimutagenic agent.

EXAMPLE 7

Hyaluronidase-inhibiting activities of polyphenols present in unripefruits

In the present Example, hyaluronidase-inhibiting activity was measuredto use it as an indicator for antiallergic activity.

Starting materials and preparation of samples

The same as used in Example 6.

Test method for hyaluronidase-inhibiting activity

Measurement of hyaluronidase-inhibiting activity was conducted based ona modification of the method described in J. Biol., vol. 250, p. 79,1975. That is, a sample solution was added to a commercial hyaluronidasesolution, and preincubation was conducted. Thereto was added a compound48/80 (histamine releasant) solution, and hyaluronidase was activated at37° C. Then, a hyaluronic acid solution was added as a substrate, and areaction was allowed to take place.

The N-acetylglucosamine produced by the above reaction was subjected tothe Elson-Morgan's procedure to develop a color. The color was measuredfor absorbance at 586 nm. Hyaluronidase-inhibiting activity (%) wascalculated using the following formula:

    hyaluronidase-inhibiting activity (%)={(C-C.sub.B)-(S-S.sub.B)}÷(C-C.sub.B) ×100

wherein S: absorbance of test solution,

C: absorbance of control wherein a buffer solution is used in place ofthe sample,

S_(B) : absorbance of blank for S (the blank contained no enzyme), and

C_(B) : absorbance of blank for C (the blank contained no sample and noenzyme).

Results

The hyaluronidase-inhibiting activities of various fruit extracts areshown in FIG. 15, and the hyaluronidase-inhibiting activities of sodiumcromoglicate and apple tannin are shown in FIG. 16.

As a result, each fruit extract and apple tannin showedhyaluronidase-inhibiting activities dependently of their concentrations.In FIG. 16, the hyaluronidase-inhibiting activity of sodium cromoglicateis shown for comparison. The IC₅₀ (the concentration at which 50%inhibition is exhibited) of sodium cromoglicate was about 0.051 mg/mlwhile the IC₅₀ of apple tannin was about 0.086 mg/ml and close to thatof sodium cromoglicate.

Thus, this polyphenol (apple tannin) contained a large amount ofcompounds inhibiting the activity of hyaluronidase (an enzyme associatedwith I type allergy). Therefore, the fruit polyphenol obtained in thepresent invention is very effective also as an antiallergic agent.

EXAMPLE 8

Glucosyltransferase-inhibiting activities of polyphenols present inunripe fruits

In the present Example were investigated the inhibitory activities offruit polyphenols to GTase [this enzyme forms insoluble glucan fromsucrose and is produced by S. sobrinus (a typical cariogenic bacteria].

Starting materials and preparation of samples

The same as used in Examples 6 and 7.

Bacterium used: Streptococcus sobrinus ATCC 33478

Preparation of GTase

GTase of S. sobrinus was prepared as follows.

S. sobrinus was cultured in a TTY medium [Agric. Biol. Chem., 54 (11),pp. 2925-2929, 1990] at 37° C. for 18 hours. Centrifugation wasconducted to remove bacterial cells and obtain a supernatant. To thesupernatant was added ammonium sulfate to 50% saturation. Then,centrifugation was conducted to recover the resulting precipitate. Theprecipitate was redissolved in a 0.05 M phosphate buffer solution (pH6.5). The resulting solution was dialyzed with the same buffer solution.The GTase in the dialyzate was purified and removed by hydroxyapatitechromatography. The GTase (capable of forming insoluble glucan) waseluted with an about 0.4 M phosphate buffer solution. TheGTase-containing elute was used for the following test.

Test method for GTase-inhibiting activity

A purified GTase solution and a sample solution were added to 1 ml of asubstrate solution [a 0.1 M phosphate buffer solution (pH 6.5)containing 2% of sucrose, 0.1% of sodium azide and 40 μM of dextranT10]. Water was added for dilution to make the total volume 2 ml. Theresulting solution was subjected to a reaction at 37° C. for 18 hours.The amount of insoluble glucan formed by the reaction was measured as aturbidity expressed by an absorbance at 550 nm, and the formation ratio(%) of insoluble glucan was calculated using the following formula:

    formation ratio (%) of insoluble glucan=(SS-SB)÷(CS-CB)×100

wherein SS: absorbance of sample,

SB: absorbance of blank for sample (the blank contained no enzyme),

CS: absorbance of control (containing no sample), and

CB: absorbance of blank for control (the blank contained no sample andno enzyme).

Results

The GTase-inhibiting activities of the polyphenol fractions of variousfruit extracts are shown in FIG. 17, and the GTase-inhibiting activitiesof individual polyphenol compounds are shown in FIG. 18. In each ofFIGS. 17 and 18, the axis of ordinate indicates the formation ratio (%)of insoluble glucan, and the formation ratio (%) in control (containingno sample) was taken as 100%. The axis of abscissa indicates the amount(volume or concentration) of sample or polyphenol added to reactionsystem.

As shown in FIG. 17, of the polyphenol fractions of various fruitextracts, particularly the polyphenol fractions of unripe apple "Fuji"and unripe pear "Niitaka" showed striking GTase-inhibiting activities.The IC₅₀ of ripe apple "Fuji" to GTase was about 50 μl while the IC₅₀ ofunripe apple "Fuji" to GTase was about 0.7 μl. Thus, the unripe applehad a GTase-inhibiting activity about 70 times higher than ripe apple.

Next, there were investigated the GTase-inhibiting activities of variouspolyphenol compounds including those present in apple. As shown in FIG.18, of the polyphenol compounds present in apple, chlorogenic acid andphloridzin each had a very low GTase-inhibiting activity. Meanwhile, ofthe catechins, epicatechin monomer had a very low GTase-inhibitingactivity but high-molecular epicatechin polymers (indicated as appletannin in FIG. 18) represented by the estimated structural formula ofFIG. 12 had a high GTase-inhibiting activity. In FIG. 18 is also shownthe result of epicatechin gallate (a typical catechin present in greentea and known as a GTase-inhibiting substance) [Agric. Biol. Chem., 5(11), pp. 2925-2929, 1990; Chem. Pharm. Bull., 38 (39), pp. 717-720,1990]. From the calculation made using FIG. 18, the IC₅₀ of epicatechingallate to GTase was about 200 ppm while that of apple tannin was about2 ppm. Thus, apple tannin had a GTase-inhibiting activity about 100times higher than epicatechin gallate. From these results, the highGTase-inhibiting activity of unripe apple "Fuji" seen in FIG. 17 isthought to be owing to the presence of apple tannin.

Thus, the fruit polyphenol obtained in the present invention containscompounds of high GTase-inhibiting activity and accordingly is veryeffective also as an anticariogenic agent.

What is claimed is:
 1. A mixture consisting essentially of polyphenolsobtained by pressing or extraction of unripe Rosaceae fruits to form ajuice or extract, treating the juice or extract with an absorbent whichselectively adsorbs the polyphenols contained in the juice or extract,eluting the polyphenols from the adsorbent with ethyl alcohol, andrecovering the mixture of polyphenols from the eluate; the mixture ofpolyphenols consisting essentially of caffeic acid, caffeic acid esters,p-coumaric acid, p-coumaric acid esters, phloretin, phloretinglycosides, quercetin, quercetin glycosides, catechin, epicatechin, andcondensed tannins.
 2. A mixture of polyphenols according to claim 1,wherein the unripe Rosaceae fruits are apples, pears or peaches.
 3. Amixture of polyphenols according to claim 1, wherein the mixture ofpolyphenols is recovered from the eluate by spray-drying.
 4. A mixtureof polyphenols according to claim 1, wherein the mixture of polyphenolsis recovered from the eluate by freeze-drying.
 5. The mixture of claim 1wherein the Rosaceae fruit used to form the juice or extract are unripeapples and the phenolic components of the mixture, catechin andepicatechin, are expressed in an HPLC chromatogram as sharp peaks at 280nm at a retention time between 4 and 20 minutes as shown in FIG.
 1. 6.The mixture of claim 1 wherein the Rosaceae fruit used to form the juiceor extract are unripe apples and the phenolic component of the mixture,caffeic acid, is expressed in an HPLC chromatogram as a sharp peak at325 nm at a retention time between 10 and 20 minutes as shown in FIG. 1.7. The mixture of claim 1 wherein the Rosaceae fruit used to form thejuice or extract are unripe apples and the phenolic component, quercetinglycoside, is expressed in an HPLC chromatogram as several small peaksat 350 nm at a retention time between 30 and 70 minutes as shown in FIG.1.